阅读说明：本技术 用于检测人免疫球蛋白基因重排的毛细管电泳试剂盒及其使用方法 (Capillary electrophoresis kit for detecting human immunoglobulin gene rearrangement and use method thereof ) 是由 陶慧卿 熊慧 谢立群 李云航 俞浩 杨建清 于 2018-06-28 设计创作，主要内容包括：本申请涉及基因检测领域,具体讲,涉及一种用于检测人免疫球蛋白基因重排毛细管电泳试剂盒及其使用方法。本申请的试剂盒包括核酸扩增试剂和对照品。本申请试剂盒采用PCR加毛细管电泳技术,具有操作简单、检测速度快、灵敏度高、检测结果直观易懂、诊断准确率高的技术优势。(The application relates to the field of gene detection, in particular to a capillary electrophoresis kit for detecting human immunoglobulin gene rearrangement and a using method thereof. The kit of the present application includes nucleic acid amplification reagents and controls. The kit adopts the PCR and capillary electrophoresis technology, and has the technical advantages of simple operation, high detection speed, high sensitivity, intuitive and understandable detection result and high diagnosis accuracy.)

1. A capillary electrophoresis kit for detecting human immunoglobulin gene rearrangement is characterized by comprising a nucleic acid amplification reagent and a reference substance;

wherein the nucleic acid amplification reagent comprises the following components:

the 5' end of the fluorescent primer is marked with a fluorescent group;

the control comprises the following components:

2. the capillary electrophoresis kit of claim 1, wherein the 5 'end of SEQ ID NO 30 is labeled with FAM, the 5' end of SEQ ID NO 31 is labeled with Tamara, the 5 'end of SEQ ID NO 32 is labeled with Hex, the 5' end of SEQ ID NO 33 is labeled with Hex, the 5 'end of SEQ ID NO 34 is labeled with Tamara, and the 5' ends of SEQ ID NO 35, SEQ ID NO 37, SEQ ID NO 39, and SEQ ID NO 41 are labeled with Rox.

3. The capillary electrophoresis kit of claim 1, wherein the concentration of each primer in the nucleic acid amplification reagent is:

the concentration of SEQ ID NO 1, SEQ ID NO 2, SEQ ID NO 3, SEQ ID NO 4, SEQ ID NO 5, SEQ ID NO 6, SEQ ID NO 7 and SEQ ID NO 30 in the immunoglobulin heavy chain A PCR reaction solution is 0.25 mu M;

the concentrations of SEQ ID NO 8, SEQ ID NO 9, SEQ ID NO 11, SEQ ID NO 12, SEQ ID NO 13, SEQ ID NO 14, SEQ ID NO 15, SEQ ID NO 16 and SEQ ID NO 30 in the immunoglobulin heavy chain B PCR reaction solution are 0.25 mu M;

the concentration of SEQ ID NO 17, 18, 19, 20, 21, 22 and 31 in the immunoglobulin heavy chain D PCR reaction solution is 0.25 mu M;

the concentrations of SEQ ID NO 23 and SEQ ID NO 28 in the immunoglobulin kappa light chain A PCR reaction solution are 0.125 mu M; the concentration of SEQ ID NO 25 is 0.25. mu.M; the concentrations of SEQ ID NO 24, 26 and 27 were 0.5. mu.M; the concentration of SEQ ID NO 32 and SEQ ID NO 33 was 0.125. mu.M;

the concentrations of SEQ ID NO 23, SEQ ID NO 25, SEQ ID NO 28 and SEQ ID NO 29 in the immunoglobulin kappa light chain B PCR reaction solution are 0.25 mu M; the concentrations of SEQ ID NO 24, 26 and 27 were 0.5. mu.M; 34 is 0.25. mu.M;

the concentration of SEQ ID NO 35 and SEQ ID NO 36 in the quality control PCR reaction solution is 0.125 mu M; the concentration of SEQ ID NO 37 and SEQ ID NO 38 is 0.25. mu.M; the concentration of SEQ ID NO 39, SEQ ID NO 40 was 0.375. mu.M; the concentration of SEQ ID NO 41 and 42 was 0.75. mu.M.

4. The capillary electrophoresis kit of claim 3, wherein the immunoglobulin heavy chain A PCR reaction solution, the immunoglobulin heavy chain B PCR reaction solution, the immunoglobulin heavy chain D PCR reaction solution, the immunoglobulin kappa light chain A PCR reaction solution, the immunoglobulin kappa light chain B PCR reaction solution, and the quality control PCR reaction solution further comprise 1.5mM of Mg²⁺And 125. mu.M dNTPs.

5. The capillary electrophoresis kit of claim 1, wherein the effective detection range of the immunoglobulin heavy chain A PCR reaction solution is 295 bp-378 bp rearranged segment of target gene FR 1-JH; the effective detection range of the immunoglobulin heavy chain B PCR reaction solution is 236 bp-307 bp rearranged segments of a target gene FR 2-JH; the effective detection range of the immunoglobulin heavy chain D PCR reaction solution is 102 bp-296 bp and 384 bp-429 bp rearranged segments of the target gene DH-JH; the effective detection range of the immunoglobulin kappa light chain A PCR reaction solution is 120 bp-160 bp, 190 bp-210 bp and 260 bp-300 bp rearrangement fragments of a target gene Vk-Jk; the effective detection range of the immunoglobulin kappa light chain B PCR reaction solution is 210 bp-250 bp, 265 bp-300 bp and 350 bp-390 bp rearrangement fragments of target genes Vk-Kde and intron-Kde.

6. The capillary electrophoresis kit of claim 1, wherein the sample for which the capillary electrophoresis kit is applied is selected from the group consisting of paraffin-embedded sections and bone marrow samples.

7. A primer for detecting human immunoglobulin gene rearrangement, wherein the sequence of the primer is:

primers shown by SEQ ID NO 1-29, 36, 38, 40 and 42; fluorescent primers shown by SEQ ID NO 30-35, SEQ ID NO 37, SEQ ID NO 39 and SEQ ID NO 41;

the 5' end of the fluorescent primer is marked with a fluorescent group.

8. The primer of claim 7, wherein the 5 'end of SEQ ID NO. 30 is labeled with FAM, the 5' end of SEQ ID NO. 31 is labeled with Tamara, the 5 'end of SEQ ID NO. 32 is labeled with Hex, the 5' end of SEQ ID NO. 33 is labeled with Hex, the 5 'end of SEQ ID NO. 34 is labeled with Tamara, and the 5' ends of SEQ ID NO. 35, SEQ ID NO. 37, SEQ ID NO. 39, and SEQ ID NO. 41 are labeled with Rox.

9. A method of using the capillary electrophoresis kit according to any of claims 1 to 6, comprising at least the steps of:

(1) preparation of amplification reagents: taking immunoglobulin heavy chain A PCR reaction liquid, immunoglobulin heavy chain B PCR reaction liquid and immunoglobulin heavy chain D PCR reaction liquid from the capillary electrophoresis kit, and respectively adding DNA polymerase liquid to obtain corresponding PCR premix;

(2) sample adding: respectively sampling from a sample DNA solution, a positive control 1 and a blank control, and respectively adding the samples into an immunoglobulin heavy chain A PCR premixed solution, an immunoglobulin heavy chain B PCR premixed solution, an immunoglobulin heavy chain D PCR premixed solution, an immunoglobulin kappa light chain A PCR premixed solution and an immunoglobulin kappa light chain B PCR premixed solution; respectively sampling from the sample DNA solution, the positive control 2 and the blank control, and respectively adding the samples into the quality control PCR premix solution;

(3) PCR amplification;

(4) capillary electrophoresis: mixing an immunoglobulin heavy chain A PCR amplification product, a quality control PCR amplification product and a capillary electrophoresis reagent, mixing an immunoglobulin heavy chain B PCR amplification product, a quality control PCR amplification product and a capillary electrophoresis reagent, mixing an immunoglobulin heavy chain D PCR amplification product, a quality control PCR amplification product and a capillary electrophoresis reagent, mixing an immunoglobulin kappa light chain A PCR amplification product, a quality control PCR amplification product and a capillary electrophoresis reagent, mixing an immunoglobulin kappa light chain B PCR amplification product, a quality control PCR amplification product and a capillary electrophoresis reagent, denaturing, cooling and performing capillary electrophoresis;

(5) the data is processed and analyzed.

Technical Field

The application relates to the field of gene detection, in particular to a capillary electrophoresis kit for detecting human immunoglobulin gene rearrangement and a using method thereof.

Background

Malignant lymphoma (malignantlymphoma) is a primary malignancy of the immune system, occurring primarily in lymphoid organs and tissues. Malignant lymphoma is divided into Hodgkin Lymphoma (HL) and non-Hodgkin lymphoma (NHL), and in China, the incidence rate of NHL is the absolute dominance (95%) of malignant lymphoma, wherein B-NHL is the majority.

The clinical diagnosis of lymphoma mainly depends on the histomorphometric examination of lymph node biopsy pathology, and since the cell proliferation in lymph nodes is very active and the cells at various differentiation stages are mixed together, the morphological diagnosis is more dependent on the working experience and clinical judgment throughout the year. Sometimes, even among the elderly pathologists, a case should be diagnosed as either reactive lymph node hyperplasia or lymphoma, often leading to disputes. Therefore, a rapid, specific and sensitive method for clinical assessment of NHL is needed.

With the development of molecular biology theories and techniques, techniques for diagnosing diseases from gene level studies and analyses are emerging and becoming mature. As early as 2001, the new classification of WHO lymphomas based on histopathology combined with immunohistochemistry and genetic analysis showed this trend.

Molecular diagnostic techniques have unique objective advantages for the diagnosis of lymphoma. The rearrangement of antigen receptor genes is often seen in B lymphocyte individuals, and the gene rearrangement products are unique in length and sequence, so that the identification of the rearrangement condition in the antigen receptor genes by using Polymerase Chain Reaction (PCR) can judge whether the detected lymphocyte populations originate from the same cell, which provides another analysis index for the diagnosis of lymphocyte malignant lesions.

The following methods are currently used for detecting immunoglobulin gene rearrangement:

the rearranged gene can be detected by Southern blot hybridization and PCR electrophoresis (PCR + agarose gel electrophoresis, PCR + capillary electrophoresis) to judge the cloning property.

Southern hybridization is a classical experimental approach in molecular biology. The basic principle is that DNA sample to be detected is fixed on a solid phase carrier and hybridized with a labeled nucleic acid probe, if the substance to be detected contains a sequence complementary with the probe, the two are combined by the base complementary principle, and the free probe is washed and then detected by self-development or other suitable technologies, so that the fragment to be detected and the relative size of the fragment to be detected are displayed. The technology is widely applied to the researches such as genetic disease detection, DNA fingerprint analysis, PCR product judgment and the like.

The detection result of the Southern blot technology is reliable, but the technology needs to prepare a probe, and the steps of Southern blotting, molecular hybridization, autoradiography and the like are complex and time-consuming, and the technology needs larger DNA, fresh or frozen tissues, has the defects of radioisotope pollution, low sensitivity (5-10 percent) and the like, and restricts the application of the technology in practical clinical pathology.

The PCR + agarose gel electrophoresis technique is an enzymatic synthesis reaction relying on DNA polymerase in the presence of template DNA, primers and four deoxyribonucleotides. DNA polymerase initiates synthesis by using a small length of double stranded DNA with single stranded DNA as a template, and forms a partial double strand by binding one or two artificially synthesized oligonucleotide primers to a complementary sequence in the single stranded DNA template. Under appropriate temperature and environment, DNA polymerase adds deoxymononucleotide to the 3 ' -OH end of the primer, and takes the deoxymononucleotide as a starting point to extend along the 5 ' → 3 ' direction of the template to synthesize a new DNA complementary strand.

The PCR and agarose gel electrophoresis technology has the advantages of high sensitivity, simplicity, convenience, rapidness, low requirement on a specimen, low cost and easy popularization. However, agarose gel electrophoresis, ethidium bromide staining and ultraviolet light are needed for observation, which wastes time and labor, and the staining agent ethidium bromide is harmful to human bodies, and the complicated experimental processes provide opportunities for pollution and false positive. And the resolution is low, about 20bp, and the misjudgment is easy.

In view of this, the present application is specifically made.

Disclosure of Invention

The invention of the present application aims to provide a kit for detecting human immunoglobulin gene rearrangement and a use method thereof.

In order to accomplish the purpose of the application, the technical scheme is as follows:

the application provides a capillary electrophoresis kit for detecting human immunoglobulin gene rearrangement, which comprises a nucleic acid amplification reagent and a reference substance;

wherein, the nucleic acid amplification reagent comprises the following components which are shown in the table 1 specifically:

TABLE 1

The 5' end of the fluorescent primer is marked with a fluorescent group;

the reference comprises the following components, specifically shown in table 2:

TABLE 2

Optionally, FAM is labeled at the 5 'end of SEQ ID NO. 30, Tamara is labeled at the 5' end of SEQ ID NO. 31, Hex is labeled at the 5 'end of SEQ ID NO. 32, Hex is labeled at the 5' end of SEQ ID NO. 33, Tamara is labeled at the 5 'end of SEQ ID NO. 34, and Rox is labeled at the 5' ends of SEQ ID NO. 35, SEQ ID NO. 37, SEQ ID NO. 39, and SEQ ID NO. 41.

Optionally, the concentration of each primer in the nucleic acid amplification reagent is:

the concentration of SEQ ID NO 1, SEQ ID NO 2, SEQ ID NO 3, SEQ ID NO 4, SEQ ID NO 5, SEQ ID NO 6, SEQ ID NO 7 and SEQ ID NO 30 in the immunoglobulin heavy chain A PCR reaction solution is 0.25 mu M; the concentrations of SEQ ID NO 8, SEQ ID NO 9, SEQ ID NO 11, SEQ ID NO 12, SEQ ID NO 13, SEQ ID NO 14, SEQ ID NO 15, SEQ ID NO 16 and SEQ ID NO 30 in the immunoglobulin heavy chain B PCR reaction solution are 0.25 mu M; the concentration of SEQ ID NO 17, 18, 19, 20, 21, 22 and 31 in the immunoglobulin heavy chain D PCR reaction solution is 0.25 mu M; the concentrations of SEQ ID NO 23 and SEQ ID NO 28 in the immunoglobulin kappa light chain A PCR reaction solution are 0.125 mu M; the concentration of SEQ ID NO 25 is 0.25. mu.M; the concentrations of SEQ ID NO 24, 26 and 27 were 0.5. mu.M; the concentration of SEQ ID NO 32 and SEQ ID NO 33 was 0.125. mu.M; the concentrations of SEQ ID NO 23, SEQ ID NO 25, SEQ ID NO 28 and SEQ ID NO 29 in the immunoglobulin kappa light chain B PCR reaction solution are 0.25 mu M; the concentrations of SEQ ID NO 24, 26 and 27 were 0.5. mu.M; 34 is 0.25. mu.M; the concentration of SEQ ID NO 35 and SEQ ID NO 36 in the quality control PCR reaction solution is 0.125 mu M; the concentration of SEQ ID NO 37 and SEQ ID NO 38 is 0.25. mu.M; the concentration of SEQ ID NO 39, SEQ ID NO 40 was 0.375. mu.M; the concentration of SEQ ID NO 41 and 42 was 0.75. mu.M.

Optionally, the immunoglobulin heavy chain a PCR reaction solution, the immunoglobulin heavy chain B PCR reaction solution, the immunoglobulin heavy chain D PCR reaction solution, the immunoglobulin kappa light chain a PCR reaction solution, the immunoglobulin kappa light chain B PCR reaction solution, and the quality control PCR reaction solution further contain 1.5mM Mg²⁺And 125. mu.M dNTPs.

Optionally, the effective detection range of the immunoglobulin heavy chain A PCR reaction solution is 295 bp-378 bp rearranged segments of a target gene FR 1-JH; the effective detection range of the immunoglobulin heavy chain B PCR reaction solution is 236 bp-307 bp rearranged segments of a target gene FR 2-JH; the effective detection range of the immunoglobulin heavy chain D PCR reaction solution is 102 bp-296 bp and 384 bp-429 bp rearranged segments of the target gene DH-JH; the effective detection range of the immunoglobulin kappa light chain A PCR reaction solution is 120 bp-160 bp, 190 bp-210 bp and 260 bp-300 bp rearrangement fragments of a target gene Vk-Jk; the effective detection range of the immunoglobulin kappa light chain B PCR reaction solution is 210 bp-250 bp, 265 bp-300 bp and 350 bp-390 bp rearrangement fragments of target genes Vk-Kde and intron-Kde.

Optionally, the sample for which the capillary electrophoresis kit is applied is selected from paraffin-embedded sections and bone marrow samples.

The application also relates to a primer for detecting human immunoglobulin gene rearrangement, which has the following sequence: primers shown by SEQ ID NO 1-29, 36, 38, 40 and 42; fluorescent primers shown by SEQ ID NO 30-35, SEQ ID NO 37, SEQ ID NO 39 and SEQ ID NO 41; the 5' end of the fluorescent primer is marked with a fluorescent group.

Optionally, FAM is labeled at the 5 'end of SEQ ID NO. 30, Tamara is labeled at the 5' end of SEQ ID NO. 31, Hex is labeled at the 5 'end of SEQ ID NO. 32, Hex is labeled at the 5' end of SEQ ID NO. 33, Tamara is labeled at the 5 'end of SEQ ID NO. 34, and Rox is labeled at the 5' ends of SEQ ID NO. 35, SEQ ID NO. 37, SEQ ID NO. 39, and SEQ ID NO. 41.

The application also relates to a using method of the capillary electrophoresis kit, which at least comprises the following steps:

(1) preparation of amplification reagents: taking immunoglobulin heavy chain A PCR reaction liquid, immunoglobulin heavy chain B PCR reaction liquid, immunoglobulin heavy chain D PCR reaction liquid, immunoglobulin kappa light chain A PCR reaction liquid, immunoglobulin kappa light chain B PCR reaction liquid and quality control PCR reaction liquid out of the capillary electrophoresis kit, and respectively adding DNA polymerase liquid to obtain corresponding PCR premix;

(2) sample adding: respectively sampling from a sample DNA solution, a positive control 1 and a blank control, and respectively adding the samples into an immunoglobulin heavy chain A PCR premixed solution, an immunoglobulin heavy chain B PCR premixed solution, an immunoglobulin heavy chain D PCR premixed solution, an immunoglobulin kappa light chain A PCR premixed solution and an immunoglobulin kappa light chain B PCR premixed solution; respectively sampling from the sample DNA solution, the positive control 2 and the blank control, and respectively adding the samples into the quality control PCR premix solution;

(3) PCR amplification;

(4) capillary electrophoresis: mixing an immunoglobulin heavy chain A PCR amplification product, a quality control PCR amplification product and a capillary electrophoresis reagent, mixing an immunoglobulin heavy chain B PCR amplification product, a quality control PCR amplification product and a capillary electrophoresis reagent, mixing an immunoglobulin heavy chain D PCR amplification product, a quality control PCR amplification product and a capillary electrophoresis reagent, mixing an immunoglobulin K light chain A PCR amplification product, a quality control PCR amplification product and a capillary electrophoresis reagent, mixing an immunoglobulin K light chain BPCR amplification product, a quality control PCR amplification product and a capillary electrophoresis reagent, cooling after denaturation, and performing capillary electrophoresis;

(5) the data is processed and analyzed.

The technical scheme of the application has at least the following beneficial effects:

the capillary electrophoresis kit of the present application employs a PCR + capillary electrophoresis technique, i.e., a gene scanning technique (GeneScan), and has the following technical advantages.

The capillary electrophoresis kit has simple operation and high detection speed, and can obtain a result in four hours by directly operating the capillary electrophoresis kit on a machine after the PCR is finished, so the capillary electrophoresis kit is simple and convenient to operate, saves time, reduces pollution and is far superior to southern blot technology and agarose gel electrophoresis.

The capillary electrophoresis kit has high sensitivity, only needs a small amount of bone marrow and paraffin to embed tissue specimens, utilizes the specific fluorescent primers, excites fluorescence through laser, and fluorescent signals are collected by CCD (charge coupled device) for analysis. Compared with southern blot technique, the sensitivity is greatly improved.

The detection result of the capillary electrophoresis kit is intuitive and easy to understand, the diagnosis accuracy is high, and the resolution of the gene scanning technology is as high as 1bp, which is far higher than that of an agarose gel electrophoresis method.

The capillary electrophoresis kit is specially designed for Chinese people, so that the detection rate of the Chinese people is obviously improved.

The present application is further illustrated with reference to specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present application.

Detailed Description

The embodiment of the application relates to a capillary electrophoresis kit for detecting human immunoglobulin gene rearrangement, which comprises immunoglobulin heavy chain genes, multiple groups of specific fluorescent primers of immunoglobulin kappa light chains, specific primers, DNA polymerase solution and the like, wherein a PCR in-vitro amplification method is adopted, fluorescent dye is marked at the 5' end of one PCR primer, a PCR product is provided with the fluorescent label during PCR amplification, then high-resolution gel electrophoresis (capillary electrophoresis) is adopted to separate amplified product fragments, the length and the fluorescent signal height of the amplified fragments are determined through a fluorescent detection system (such as an ABI sequencer), and whether a cell population with clone rearrangement exists in a detected sample or not is judged.

PCR-based assays are often used to identify clonal B cell populations. Due to the polymorphism of antigen receptor genes (which make up a heterogeneous population), it is difficult to amplify all conserved regions where V-J rearrangement occurs with only one set of primers, and N-terminal diversity and somatic mutations make DNA sequence analysis of the regions more complicated. Thus, multiple primers targeting each region are needed to identify the cell population in which clonal rearrangement occurs. The nucleic acid amplification reagent of the capillary electrophoresis kit of the embodiment of the application, namely the immunoglobulin heavy chain A is used for amplifying the FR1 region and the conserved J region in the IGH variable region, the immunoglobulin heavy chain B is used for amplifying the FR2 region and the conserved J region in the IGH variable region, and the immunoglobulin heavy chain D is used for amplifying the D region and the J region. Immunoglobulin kappa light chain A amplifies IGK variable region (V) and junction region (J), and immunoglobulin kappa light chain B detects V region, Jk-Ck intron region, and Kde region. These conserved regions are mainly located on both sides of the V-J region of the complementarity determining region (CDR3) where gene rearrangement occurs during B cell maturation. The rearranged antigen receptor genes are primarily B cell immunoglobulin heavy and light chains, and each B cell V-J rearrangement is unique in length and sequence. Thus, the expected amplified fragments resulting from the amplification of the V-J region from a normal or polyclonal population of cells appear as bell-shaped curves (Gaussian distributions) on the capillary electrophoresis plot. The gaussian distribution indicates the presence of a heterogeneous population of cells undergoing V-J rearrangement in the sample being tested (in some cases, no corresponding product is produced if there is no lymphocyte DNA). Amplification of sample DNA containing a clonal population often shows 1 or 2 distinct amplification peaks in a reduced polyclonal background. The capillary electrophoresis kit is specially designed for Chinese people, so that the detection rate of the Chinese people is obviously improved.

Specifically, the capillary electrophoresis kit of the embodiment of the present application is specifically shown in table 3:

TABLE 3

Specifically, the nucleotide sequence of the primer is specifically shown in table 4:

table 4:

specifically, the nucleotide sequence of the plasmid DNA is specifically shown in table 5:

TABLE 5

Optionally, the concentration of each primer in the nucleic acid amplification reagent is:

the concentrations of SEQ ID NO 1, SEQ ID NO 2, SEQ ID NO 3, SEQ ID NO 4, SEQ ID NO 5, SEQ ID NO 6, SEQ ID NO 7 and SEQ ID NO 30 in the immunoglobulin heavy chain A PCR reaction solution were 0.25. mu.M; the concentrations of SEQ ID NO 8, SEQ ID NO 9, SEQ ID NO 11, SEQ ID NO 12, SEQ ID NO 13, SEQ ID NO 14, SEQ ID NO 15, SEQ ID NO 16 and SEQ ID NO 30 in the immunoglobulin heavy chain B PCR reaction solution were 0.25. mu.M; the concentrations of SEQ ID NO 17, 18, 19, 20, 21, 22 and 31 in the immunoglobulin heavy chain D PCR reaction solution were 0.25. mu.M; the concentrations of SEQ ID NO 23 and SEQ ID NO 28 in the immunoglobulin kappa light chain A PCR reaction solution were 0.125. mu.M; the concentration of SEQ ID NO 25 is 0.25. mu.M; the concentrations of SEQ ID NO. 24, SEQ ID NO. 26 and SEQ ID NO. 27 were 0.5. mu.M; the concentration of SEQ ID NO 32 and SEQ ID NO 33 was 0.125. mu.M; the concentrations of SEQ ID NO 23, SEQ ID NO 25, SEQ ID NO 28, and SEQ ID NO 29 in the immunoglobulin kappa light chain B PCR reaction solution were 0.25. mu.M; the concentrations of SEQ ID NO 24, 26 and 27 were 0.5. mu.M; 34 is 0.25. mu.M; the concentration of SEQ ID NO 35 and SEQ ID NO 36 in the quality control PCR reaction solution is 0.125 mu M; the concentration of SEQ ID NO 37 and SEQ ID NO 38 is 0.25. mu.M; the concentration of SEQ ID NO 39, SEQ ID NO 40 was 0.375. mu.M; the concentration of SEQ ID NO 41 and 42 was 0.75. mu.M.

Because the capillary electrophoresis kit is a multiplex PCR, amplification products with different sizes exist in 1 reaction tube, and if the concentration of the primers is not optimized, larger fragments are not easy to be amplified, and the omission factor is easy to cause. In this regard, the present application optimizes the concentration of the primer, thereby significantly improving the sensitivity of the reagent.

Optionally, the effective detection range of the immunoglobulin heavy chain A PCR reaction solution is 295 bp-378 bp rearranged segments of the target gene FR 1-JH; the effective detection range of the immunoglobulin heavy chain B PCR reaction solution is 236 bp-307 bp rearranged segments of the target gene FR 2-JH; the effective detection range of the immunoglobulin heavy chain D PCR reaction solution is 102 bp-296 bp and 384 bp-429 bp rearranged segments of the target gene DH-JH; the effective detection range of the immunoglobulin kappa light chain A PCR reaction solution is 120 bp-160 bp, 190 bp-210 bp and 260 bp-300 bp rearrangement fragments of a target gene Vk-Jk; the effective detection range of the immunoglobulin kappa light chain B PCR reaction solution is 210 bp-250 bp, 265 bp-300 bp and 350 bp-390 bp rearranged fragments of target genes Vk-Kde and intron-Kde. The capillary electrophoresis kit provided by the embodiment of the application is specially used for detecting the gene rearrangement of Chinese population, and the size range of the rearranged fragments is determined, so that the detection rate of the gene rearrangement of Chinese population can be improved.

Optionally, the capillary electrophoresis kit of the embodiment of the present application may further include a capillary electrophoresis reagent, and the capillary electrophoresis reagent includes a standard molecular weight internal standard and a DNA product denaturant. Preferably, the standard molecular weight internal standard is selected from the LIZ-500 molecular weight internal standards and the DNA product denaturant is selected from the group consisting of highly deionized formamide.

Optionally, the capillary electrophoresis kit of the embodiment of the present application further includes a DNA extraction kit.

Optionally, the concentration of the sample DNA prepared by the DNA extraction kit is 50 ng/mul-100 ng/mul.

The application method of the capillary electrophoresis kit in the embodiment of the application comprises the following steps:

1. in each test, the positive control and the blank control in the capillary electrophoresis kit must be simultaneously subjected to PCR amplification.

2. Preparation of amplification reagents:

and taking out each PCR reaction solution (immunoglobulin heavy chain A reaction solution, immunoglobulin heavy chain B reaction solution, immunoglobulin heavy chain D reaction solution, immunoglobulin kappa light chain A reaction solution, immunoglobulin kappa light chain B reaction solution and quality control PCR reaction solution) and DNA polymerase solution from the capillary electrophoresis kit, melting at room temperature, shaking, mixing uniformly, and centrifuging at 2000rpm for 10 s.

The reaction systems were prepared as follows: mu.l of PCR reaction solution and 1. mu.l of DNA polymerase solution were mixed (note that the number of PCR tubes should be the sum of the number of samples and 2 controls, wherein the blank control and the positive control 2 were detected in the quality control PCR reaction solution, and the blank control and the positive control 1 were detected in the remaining PCR reaction solutions).

Mu.l of the mixed reaction solution was added to each PCR reaction tube by a micro-applicator and stored at 4 ℃.

3. Sample adding: mu.l of sample DNA solution, positive control and blank control were added to the centrifuge tube containing the PCR premix, the tube cap was closed, and the mixture was centrifuged rapidly for 10 seconds and transferred to the detection zone.

PCR amplification: the reaction system was 20. mu.l. The PCR amplification conditions on the PCR amplification apparatus are set as shown in Table 6:

table 6:

5. capillary electrophoresis:

for the same sample, positive control or blank control, reagents were configured as follows:

mu.l immunoglobulin heavy chain A PCR amplification product + 0.5. mu.l quality control PCR amplification product + 0.3. mu.l LIZ500+ 10. mu.l HIDI;

mu.l immunoglobulin heavy chain B PCR amplification product + 0.5. mu.l quality control PCR amplification product + 0.3. mu.l LIZ500+ 10. mu.l HIDI;

and the like, configuring PCR amplification products of the immunoglobulin heavy chain D, the immunoglobulin kappa light chain A and the immunoglobulin kappa light chain B.

Denaturation at 95 ℃ for 3 min, immediately followed by cooling on ice for 3 min, and immediately followed by capillary electrophoresis on a 3130XL or 3500DX gene analyzer.

The computer automatically processes and analyzes the data.

Note that: if the fluorescence signal intensity of the PCR product exceeds the highest detection limit which can be collected by a sequencer, the PCR product can be diluted by a proper amount and then put on a computer, and the amplification product of the quality control PCR reaction solution and the amplification products of other reaction solutions are diluted by the same times.

[ Positive judgment value ]

a. Collecting 1 or 2 obvious amplification peaks with single size in an effective range from any 1 tube of reaction solution except the quality control PCR reaction solution, and judging that the sample has the following characteristics that the fluorescence signal height of the amplification peaks meets one of the conditions in the table 7: the result of the immunoglobulin gene clonality rearrangement detection is positive, which indicates that a clone cell population exists.

TABLE 7

Master Mix	Peak/peak fluorescence signal	Secondary peak/tertiary peak fluorescence signal
			Immune ballProtein heavy chain A	>3	/
Immunoglobulin heavy chain B	>3	/
			Immunoglobulin heavy chain D	>3	/
Immunoglobulin kappa light chain A	>7	>4
			Immunoglobulin kappa light chain B	>5	>3

b. If a is not satisfied

(1) If ROX fluorescent signals are not collected in the position of 100, 200, 299 and 394bp in the quality control PCR reaction solution of the sample, the sample needs to be re-extracted; if there is no fluorescence signal, then the sample is judged to be: "the amount or quality of DNA used for detection was not satisfactory and could not be judged".

(2) If ROX fluorescence signals are collected at 100, 200, 299 and 394bp positions in the quality control PCR reaction solution of the sample:

judging that the sample does not collect a fluorescence signal or collect a continuous fluorescence signal in the effective range of other reaction solutions, and judging that the fluorescence signal is represented by a bell-shaped curve: "the result of the immunoglobulin gene clonal rearrangement assay is negative or below the detection limit".

② on the contrary, it is recommended to re-detect the tube reaction solution. After re-detection, if the experimental result meets a, the result of the immunoglobulin gene clonality rearrangement detection is judged to be positive, which indicates that a clonal cell population exists; if the experimental result can not satisfy a, judging that the immunoglobulin gene clonality rearrangement detection result is negative or lower than the detection limit.

[ interpretation of test results ]

1. The experimental results should meet the following validity determinations:

a. blank control: no fluorescent signal other than the primer peak was collected.

b. Positive control 1: the other PCR reaction solutions except the quality control PCR reaction solution are in an effective detection range, discontinuous fluorescence signals are collected, obvious amplification peaks are shown, and the amplification peaks are effective when appearing on the positions shown in the table 8.

TABLE 8

c. Positive control 2: ROX (red) fluorescence signals were collected from the quality control PCR reaction solution at positions of 100, 200, 299, and 394 bp.

Remarking: the position error of the fluorescence signal is less than 3 bp.

2. There may be an amplification band of 345bp outside the effective detection range in the immunoglobulin heavy chain D, and the amplification band is a genomic DNA amplification product without concern.

3. And (3) amplification peak: fluorescence signal peak significantly above polyclonal background.